Estudios sobre la infección por Teladorsagia circumcincta en ovinos de raza churra: criterios y métodos para la identificación de animales resistentes

149 p.En el presente trabajo de Tesis Doctoral hemos pretendido establecer criterios y métodos de identificación de animales resistentes a la infección por Teladorsagia circumcincta en ovinos de raza Churra seleccionados en función de parámetros parasitológicos e inmunitarios. Durante el desarrollo del trabajo se han intentado establecer los siguientes aspectos: 1. Demostración de la relación existente entre el título de inmunoglobulina A tras una infección producida por T. circumcincta y la resistencia a la infección en ovejas de raza Churra. La resistencia a la infección se demostró mediante diversos parámetros: desarrollo del parásito en el hospedador; excreción de huevos con las heces; porcentaje de eosinófilos en sangre. 2. Influencia de la nutrición sobre la resistencia a la infección en ovejas Churras infectadas experimentalmente por T. circumcincta. La influencia de la proteína en la dieta sobre el desarrollo de la inmunidad se determinó experimentalmente administrando a los animales una dieta hipoproteica. 3. Desarrollo de una proteína recombinante con fines diagnósticos de la infección por T. circumcincta. El diagnóstico se realiza por medio de la detección de la inmunoglobulina A en ovejas resistentes y susceptibles a la infección. La finalidad es mejorar una prueba de ELISA a través de la expresión de un antígeno proteico del nematodo. 4. Comparación de dos técnicas de detección de animales resistentes a la infección por T. circumcincta. Mediante la técnica del ELISA se comparó el título de inmunoglobulina A frente antígeno somático de T. circumcincta o bien frente a un fragmento de la proteína recombinante disulfuro isomerasa en sangre, secreciones nasales y saliv

Loop-mediated isothermal amplification (LAMP) assay for the diagnosis of fasciolosis in sheep and its application under field conditions

[EN] Background: Loop-mediated isothermal amplification (LAMP) is a very specific, efficient, and rapid gene amplification procedure in which the reaction can run at a constant temperature. In the current study we have developed a LAMP assay to improve the diagnosis of Fasciola spp. in the faeces of sheep. Findings: After the optimisation of the LAMP assay we have shown similar results between this technique and the standard PCR using the outer primers of the LAMP reaction. In both cases the limit of detection was 10 pg; also, the diagnosis of fasciolosis was confirmed during the first week post-infection in experimental infected sheep by both techniques. In eight naturally infected sheep, the infection with F. hepatica was confirmed in all animals before a treatment with triclabendazole and on day 30 post treatment in two sheep using the LAMP assay; however, when we carried out the standard PCR with the outer primers, the results before treatment were the same but on day 30 post-treatment the infection was only confirmed in one out of the two sheep. On the other hand, the standard PCR took around 3 h to obtain a result, comparing with 1 h and 10 min for the LAMP assay. Conclusions: The LAMP assay described here could be a good alternative to conventional diagnostic methods to detect F. hepatica in faeces since it solves the drawbacks of the standard PCRSIWe thank Drs Manga-González and González-Lanza (Instituto de Ganadería de Montaña, CSIC-ULE, Spain) for providing DNA samples of D. dendriticum and C. daubneyi. This study has been funded by the national project INIA (Instituto Nacional de Investigaciones Agrarias: RTA2010-00094-C03-02), Ministry of Economy and Competitiveness (Ministerio de Economia y Competitividad), and also co-funded by the European Regional Development Fund (Fondos Feder

Αvά y κατά EN HERODOTO. ANALISIS ESTRUCTURAL

Il s' agit d' un essai de description des prépositions άνά y κατά dans les livres I et II de l' 'Histoire' d' Hérodote, leurs opositions et leurs possibles neutralisations, pour délimiter finalement leur valeur sémantique.Il s' agit d' un essai de description des prépositions άνά y κατά dans les livres I et II de l' 'Histoire' d' Hérodote, leurs opositions et leurs possibles neutralisations, pour délimiter finalement leur valeur sémantique

Social innovation thought women`s collaboration networks for sustainable development: a study of local network in Honduras

El propósito de este artículo es proporcionar un análisis de cómo las redes de colaboración de mujeres en Honduras, dentro y fuera de las empresas, tienen impacto en la innovación social, entendida -de momento- como ideas buenas para el mundo-, el desarrollo sostenible y la solidaridad. Basada en la propia experiencia y estudio de campo de la autora, en este artículo evidencia la maximización del capital social (entendido como incremento del valor) que estas redes generan y su impacto de manera colectiva e individual en las mujeres que las conformanThe purpose of this article is to provide an analytical approach of how women collaboration networks in Honduras (lean in circle and others), inside and out of the companies, have an impact on social innovation, social development, and solidarity. Base on the experience and field research of the author, this article acknowledges how these networks maximize social capital and has an impact on individual and collective level for the wome

Zoonotic implications of onchocerca species on human health

The genus Onchocerca includes several species associated with ungulates as hosts, although some have been identified in canids, felids, and humans. Onchocerca species have a wide geographical distribution, and the disease they produce, onchocerciasis, is generally seen in adult individuals because of its large prepatency period. In recent years, Onchocerca species infecting animals have been found as subcutaneous nodules or invading the ocular tissues of humans; the species involved are O. lupi, O. dewittei japonica, O. jakutensis, O. gutturosa, and O. cervicalis. These findings generally involve immature adult female worms, with no evidence of being fertile. However, a few cases with fertile O. lupi, O. dewittei japonica, and O. jakutensis worms have been identified recently in humans. These are relevant because they indicate that the parasite's life cycle was completed in the new host-humans. In this work, we discuss the establishment of zoonotic Onchocerca infections in humans, and the possibility of these infections to produce symptoms similar to human onchocerciasis, such as dermatitis, ocular damage, and epilepsy. Zoonotic onchocerciasis is thought to be an emerging human parasitic disease, with the need to take measures such as One Health Strategies, in order to identify and control new cases in humans

Quantification of resistant alleles in the β-tubulin gene of field strains of gastrointestinal nematodes and their relation with the faecal egg count reduction test

© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.[EN]Background: Benzimidazole (BZ) resistance in gastrointestinal nematodes is associated with a single nucleotide polymorphism (SNP) at codons 167, 198 and 200 in the isotype 1 of beta-tubulin gene although in some species these SNPs have also been associated with resistance to macrocyclic lactones. In the present study we compared the levels of resistance in Teladorsagia circumcincta and Trichostrongylus colubriformis by means of the faecal egg reduction test (FECRT) and the percentage of resistant alleles obtained after pyrosequencing. The study was conducted in 10 naturally infected sheep flocks. Each flock was divided into three groups: i) group treated with albendazole (ABZ); ii) group treated with ivermectin (IVM); iii) untreated group. The number of eggs excreted per gram of faeces was estimated at day 0 and 14 post-treatment. Results: Resistance to ABZ was observed in 12.5% (1/8) of the flocks and to IVM in 44.4% (4/9) of them. One flock was resistant to both drugs according to FECRT. Coprocultures were performed at the same dates to collect L3 for DNA extraction from pooled larvae and to determine the resistant allele frequencies by pyrosequencing analysis. In T. circumcincta, SNPs were not found at any of the three codons before treatment; after the administration of ABZ, SNPs were present only in two different flocks, one of them with a frequency of 23.8% at SNP 167, and the other 13.2% % at SNP 198. In relation to T. colubriformis, we found the SNP200 before treatment in 33.3% (3/9) of the flocks with values between 48.5 and 87.8%. After treatment with ABZ and IVM, the prevalence of this SNP increased to 75 and 100% of the flocks, with a mean frequency of 95.1% and 82.6%, respectively. Conclusion: The frequencies observed for SNP200 in T. colubriformis indicate that the presence of resistance is more common than revealed by the FECRT.SIThis study has been funded by the national project INIA (Instituto Nacional de Investigaciones Agrarias: RTA2013-00064-C02-02) of the Ministry of Economy and Competitiveness (Ministerio de Economía y Competitividad), the European Regional Development Fund (Fondos Feder), the Spanish “Ramón y Cajal” Programme of the Ministry of Economy and Competitiveness (MMV, RYC-2015-18368), and the Cooperativa Bajo Duero, COBADU. We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI). The authors would like to thank all animal owners for their willingness to collaborate in the study

Exploring the mechanisms of resistance to Teladorsagia circumcincta infection in sheep through transcriptome analysis of abomasal mucosa and abomasal lymph nodes

8 p.The present study exploited the RNA-seq technology to analyze the transcriptome of target tissues affected by the Teladorsagia circumcincta infection in two groups of adult ewes showing different statuses against gastrointestinal nematode (GIN) infection with the aim of identifying genes linked to GIN infection resistance in sheep. For this, based on the accumulated faecal egg count of 18 adult Churra ewes subjected to a first experimental infection with T. circumcincta, six ewes were classified as resistant and six others as susceptible to the infection. These 12 animals were dewormed and infected again. After humanitarian sacrifice of these 12 animals at day 7 post-infection, RNA samples were obtained from abomasal mucosa and lymph node tissues and RNA-Seq datasets were generated using an Illumina HiSeq 2000 sequencer. The distribution of the genes based on their expression level were very similar among the two different tissues and conditions. The differential expression analysis performed with two software (DESeq and EdgeR) only identified common differentially expressed genes (DEGs), a total of 106, in the lymph node samples which were considered as GIN-activated. The enrichment analysis performed for these GIN-activated genes identified some pathways related to cytokine-mediated immune response and the PPARG signaling pathway as well as disease terms related to inflammation and gastro-intestinal diseases as enriched. A systematic comparison with the results of previous studies confirmed the involvement of genes such as ITLN2, CLAC1 and galectins, in the immune mechanism activated against T. circumcincta in resistant sheep. Financiado por la Junta de Castilla y León (Conserjería de Educación) y Fondo Social Europeo (Ref. LE248U14). Ejecución: Universidad de León. Dpto. Producción Animal. Investigador Principal: Dr. Beatriz Gutierrez GilS

Variant discovery in genes identified as differentially expressed genes between the abomasal lymph node transcriptome of resistant and susceptible adult sheep to teladorsagia circumcincta infection

Gastrointestinal nematode infections are one of the major health issues facing grazing sheep populations and it incurs on major economic losses for sheep breeders. The resistance/susceptibility trait appears to be a highly complex trait (Behnke et al. 2003; Dominik 2005). In sheep resistance to nematode infection shows a moderate level of heritability (range 0.3-0.6) (Stear et al. 2001). Several QTL mapping studies have tried to identify genomic regions and mutations that influence resistance to nematode infection (Atlija et al. 2016; Coltman et al. 2001; Gutiérrez-Gil et al. 2009; Sayers et al. 2005), although the detection of causal mutations for this trait is still a challenge for the research community. The recently available RNA-seq technology provides the opportunity to extract highthroughput transcriptome data from a specific tissue to perform gene quantification, differential gene expression and detection of variants (SNPs and indels), which could be assessed as potential causal mutations (Hudson, Dalrymple, and Reverter 2012). A previous study of our research group has identified a list of 106 differential expression genes (DEGs) based on RNA-Seq dataset obtained from the abomasal lymph nodes of 12 adult sheep, previously classified as resistant or susceptible to GIN infection based on an artificial infection with T. circumcincta larvae Chitneedi et al. (2018). In the present study we present a detailed study of the variants mapping within the list of DEGs previously reported in that study. Thus, the present study provides a list of functionally relevant variants that could underlie the genetic control of resistance/susceptibility to T. circumcincta in adult sheep. Financiado por la Junta de Castilla y León (Conserjería de Educación) y Fondo Social Europeo (Ref. LE248U14). Ejecución: Universidad de León. Dpto. Producción Animal. Investigador Principal: Dr. Beatriz Gutierrez Gi

Identification of Regulatory Functions of LncRNAs Associated With T. circumcincta Infection in Adult Sheep

[EN] Several recent studies have demonstrated the role of long non-coding RNAs (lncRNAs) in regulating the defense mechanism against parasite infections, but no studies are available that investigated their relevance for immune response to nematode infection in sheep. Thus, the aim of the current study was to (i) detect putative lncRNAs that are expressed in the abomasal lymph node of adult sheep after an experimental infection with the gastrointestinal nematode (GIN) Teladorsagia circumcincta and (ii) to elucidate their potential functional role associated with the differential host immune response. We hypothesized that putative lncRNAs differentially expressed (DE) between samples from animals that differ in resistance to infection may play a significant regulatory role in response to nematode infection in adult sheep. To obtain further support for our hypothesis, we performed co-expression and functional gene enrichment analyses with the differentially expressed lncRNAs (DE lncRNAs). In a conservative approach, we included for this predictive analysis only those lncRNAs that are confirmed and supported by documentation of expression in gastrointestinal tissues in the current sheep gene atlas. We identified 9,105 putative lncRNA transcripts corresponding to 7,124 gene loci. Of these, 457 were differentially expressed lncRNA loci (DELs) with 683 lncRNA transcripts. Based on a gene co-expression analysis via weighted gene co-expression network analysis, 12 gene network modules (GNMs) were found significantly correlated with at least one of 10 selected target DE lncRNAs. Based on the principle of “guilt-by-association,” the DE genes from each of the three most significantly correlated GNMs were subjected to a gene enrichment analysis. The significant pathways associated with DE lncRNAs included ERK5 Signaling, SAPK/JNK Signaling, RhoGDI Signaling, EIF2 Signaling, Regulation of eIF4 and p70S6K Signaling and Oxidative Phosphorylation pathways. They belong to signaling pathway categories like Cellular Growth, Proliferation and Development, Cellular Stress and Injury, Intracellular and Second Messenger Signaling and Apoptosis. Overall, this lncRNA study conducted in adult sheep after GIN infection provided first insights into the potential functional role of lncRNAs in the differential host response to nematode infectionSIFinancial support for the experimental work of this project was received from the LE248U14 project of the Junta de Castilla and León regional government, whereas the storage and processing of the generated sequencing datasets have been funded by the RTI2018-093535-B-I00 project from the Spanish Ministry of Science and Innovation (MICINN). PC was funded by a Short- Term Scientific Missions of the Functional Annotation of Animal Genomes-European network (FAANG-Europe) COST Action CA15112 to do a short research stay at Leibniz Institute for Farm Animal Biology (FBN). MM-V was also funded by the “Ramón y Cajal” Programme (RYC-2015-18368) from MICIN

Preservation of Quercus robur germplasm by cryostorage of embryogenic cultures derived from mature trees and rapd analysis of genetic stability

This study reports on the cryostorage of embryogenic lines derived from selected mature Quercus robur trees, following application of the PVS2-vitrification based procedure. In seven oak genotypes, embryo recovery levels ranging from 57-92% were obtained when 4-6 mg embryo clumps were precultured for 3 days on 0.3 M sucrose basal medium, treated with PVS2 solution for 60 min at 24ºC, and then immersed in liquid nitrogen (LN). Embryos of six out of seven lines were cryostored for one week and one year and used to evaluate cryopreservation tolerance, germination ability and to assess genetic fidelity by random amplified polymorphic DNA (RAPD) markers. There were no significant differences between the recovery frequencies of samples retrieved from LN after 1 week and 1 year of cryostorage. In five out of six lines, RAPD profiles of cryopreserved somatic embryos and regenerated plantlets were identical to those of the controls. Although polymorphisms were detected in only one cryostored embryo of one genotype, no genetic instability was found in the regenerated plantlets. This methodology appears to be suitable for long-term storage of this valuable germplasm, as the recovered plantlets were found to be genetically stable.Xunta de Galicia project: PGIDIT03RFO40001PR MEC (Spain) project: AGL2006-01387/FORPeer reviewe